Journal: Scientific Reports

Article Title: Freeform micropatterning of living cells into cell culture medium using direct inkjet printing

doi: 10.1038/s41598-017-14726-w

Figure Lengend Snippet: Demonstration of various cell patterns that were directly printed into culture medium. ( a ) Printed cell migration assay model with 1-mm and 0.5-mm gaps. Images were captured at 2 hours and 48 hours after printing. Scale bar: 500 µm. ( b–e ) Fluorescent images of red-, green-, and blue-labeled cell patterns. Cells were pre-labeled and printed in patterns. Scale bar: 1 mm. ( f ) Printed zigzag pattern using a heterotypic co-culture model of NIH3T3 and HEK293A cells. Scale bar: 1 mm.

Article Snippet: Mouse NIH3T3 fibroblasts and HEK293A human embryonic kidney cells were provided by Prof. Kyungtae Kim, POSTECH, Korea.

Techniques: Cell Migration Assay, Labeling, Co-Culture Assay

Journal: Communications Biology

Article Title: Scanning single molecule localization microscopy (scanSMLM) for super-resolution volume imaging

doi: 10.1038/s42003-023-05364-2

Figure Lengend Snippet: a Fluorescence image of a typical NIH3T3 cells transfected with Dendra2-Actin plasmid DNA. b A small section of the cell is imaged using scanSMLM and 3D super-resolved volume is reconstructed, where colormap represents the depth. c Few 3D sections are displayed that show Actin filaments at different depths in a single cell. d , e Volume views along with the diagonal views (along lines Q1, Q2 and Q3) showing the arrangement of single Dendra2-Actin molecules. Scale = 1 μm. Additional details can be found in Supplementary note .

Article Snippet: NIH3T3 mouse fibroblast cell line is a generous gift from Prof Deepak Nair (Centre for Neuroscience, Indian Institute of Science, Bangalore, India).

Techniques: Fluorescence, Transfection, Plasmid Preparation

Journal: Communications Biology

Article Title: Scanning single molecule localization microscopy (scanSMLM) for super-resolution volume imaging

doi: 10.1038/s42003-023-05364-2

Figure Lengend Snippet: a A high-resolution fluorescence image of mitochondria in a NIH3T3 cell (transfected with mEos-Tom20 plasmid DNA). b A section of the mitochondrial network is super-resolved and the volume is reconstructed. The depth is represented by colormap (see colorbar scale). c Few chosen single mitochondria (R1, R2, R3) are resolved that shows the distribution of single molecules (Meos-Tom20). d The corresponding line intensity plots suggests the distribution of single molecules on mitochondria (L1, L2, L3). e , f Volume views and the corresponding diagonal views (along lines, Q1, Q2, Q3) display the organization of single molecules across cell depths in a single mitochondria. Additional details can be found in Supplementary note .

Article Snippet: NIH3T3 mouse fibroblast cell line is a generous gift from Prof Deepak Nair (Centre for Neuroscience, Indian Institute of Science, Bangalore, India).

Techniques: Fluorescence, Transfection, Plasmid Preparation

Journal: Communications Biology

Article Title: Scanning single molecule localization microscopy (scanSMLM) for super-resolution volume imaging

doi: 10.1038/s42003-023-05364-2

Figure Lengend Snippet: a Cluster map of the distributed HA molecules in a transfected NIH3T3 cell (of few chosen planes at 500 nm, 2500 nm and 4500 nm depth) for both cyclic and conventional scanning. DBSCAN algorithm is used to analyze clusters (with parameters radius ϵ = 114 nm and minimum molecules of 35 for it to be designated as a cluster). Analysis show aggregated HA molecules throughout the cell volume. b Volume image of a cell displaying 3D distribution of HA molecules. In addition, diagonal sectional view of the 3D cluster indicates the local density of clusters across the cell volume. Few enlarged section of a single cluster are also displayed along with orthogonal sectional views (along XY, YZ, XZ) of a specific region. Corresponding intensity plots indicate average HA cluster size of ≈ 240 nm (see, orange arrow) and typical single molecule size (localization precision) of 26.2 nm (see, blue arrow). c Estimated biophysical parameters (average cluster area, density and # molecules per cluster) related to local cell physiology indicates that most of the clusters are spread over a distance of 200 nm with an average density of about 1000#mol./μm 2 . In addition, estimated from the cell volume shows the clustered molecule fraction of ~42.77, indicating that 42.77% of the total HA molecules are clustered, and rest remains unclustered, post 24 hrs of transfection. The clustered area fraction (which is the ratio of clustered area to the total cell area) is found be about 0.981%, indicating a < 1% area is occupied by clusters. These parameters are critical for understanding HA dynamics and help determine underlying biophysical processes. More details can be found in Supplementary note and Supplementary note .

Article Snippet: NIH3T3 mouse fibroblast cell line is a generous gift from Prof Deepak Nair (Centre for Neuroscience, Indian Institute of Science, Bangalore, India).

Techniques: Transfection

Journal: Communications Biology

Article Title: Scanning single molecule localization microscopy (scanSMLM) for super-resolution volume imaging

doi: 10.1038/s42003-023-05364-2

Figure Lengend Snippet: a A cartoon of 3D cell along with real fluorescence image of the transfected NIH3T3 cell. b Cyclic scanning for entire cell volume along with reconstructed super-resolved images ( P 1 to P M ). c Super-resolved images of a few sample planes (plane 1, plane 5, and plane 10) reconstructed from raw recorded data acquired using cyclic and conventional scanning scheme. d The corresponding mean localization precision of all the reconstructed images (plane 1–10). e Plane-wise photobleaching study showing the decrease of fluorescence with time for both cyclic and conventional scan. f Signal-to-background ratio (SBR) for cyclic and conventional scan. g Change in SBR during cyclic scan. h The change in localization density with time. i Amplitude ratio versus time for cyclic scan. Scale = 1 μm. Additional details can be found in Supplementary note .

Article Snippet: NIH3T3 mouse fibroblast cell line is a generous gift from Prof Deepak Nair (Centre for Neuroscience, Indian Institute of Science, Bangalore, India).

Techniques: Fluorescence, Transfection

Journal: Scientific Reports

Article Title: Murine neonatal cardiac regeneration depends on Insulin-like growth factor 1 receptor signaling

doi: 10.1038/s41598-024-72783-4

Figure Lengend Snippet: Efficient IGF1R knockdown in the neonatal heart. A Time course of Igf1r mRNA expression in vivo in whole hearts during postnatal (P) days 1 to 7, analyzed by qPCR. qPCR was performed in biological triplicates. B In vitro KD efficiency of the Renilla control, and the three indicated Igf1r KD hairpins used for in vivo and in vitro experiments. Mouse embryonic fibroblasts (MEFs) expressing a dTomato reporter tagged with target sites of the probed shRNAmir were transduced with the indicated shRNAs. Note that “expression” refers to the expression of the dTomato reporter tagged with the target site for the responding shRNAmirs. Therefore, the Ren.713 hairpin reduced expression of the reporter tagged with the Ren target site, not with the Igf1r target sites. d = day. C Immunoblotting of in vitro IGF1R KD efficiency using the indicated hairpins, the Renilla control hairpin delivered by retroviruses, and the empty retrovirus control, tested in NIH3T3 cells. Cells were harvested on day 4 after 1 st infection. GAPDH protein expression is shown as a loading control. D In vitro Igf1r knockdown efficiency of the respective siRNAs and controls in neonatal mouse cardiomyocytes determined by qPCR. The target gene expression levels were normalized to the house keeping gene TATA-binding protein (TBP). E Representative Western blot of in vitro Igf1r knockdown efficiency of the respective siRNA and controls in neonatal mouse cardiomyocytes. For Western blotting, two runs of neonatal mouse cardiomyocyte cell culture with four cell culture wells per siRNA were performed; four cell culture wells were pooled per Western blot sample. Graph indicates mean relative signaling intensity of IGF1R relative to GAPDH ± SD, normalized to Ren.713. F In vivo luciferase imaging to confirm recombinant adeno-associated virus (rAAV) infection of the heart. The upper panel shows the plasmid sequence of the rAAV used, containing a luciferase as a reporter gene under the control of a cardiomyocyte specific troponin T promoter. The lower panel shows two mice after injection of the rAAVs on the left and a control mouse on the right into which no rAAVs were injected. Color represents the light emitted by the luciferase. While there is some leakiness of the reporter in the liver, constructs are expressed exclusively in the hearts of the mice. ITR = Inverted terminal repeat. TropT = Cardiomyocyte specific troponin T promoter. Luc2 = Firefly luciferase. MCS = Multiple cloning site. PolA = Poly-A tail. G Representative images of immunohistochemical staining of hearts to confirm in vivo plasmid expression. The upper panel shows the plasmid sequence containing GFP as a reporter gene, under the control of a cardiomyocyte specific troponin T promoter. The lower panels show the sections stained for GFP expression. Hairpins were subcloned into the multiple cloning site (MCS). The panels on the left show a negative control heart, the panels on the right represent a heart 5 days after injection of the rAAVs constructs. Brown marks GFP. Blue represents counterstaining with Hematoxylin. Areas chosen for magnification are marked with a black box. The red triangle marks the aortic valve, the orange triangle the aorta. Both do not express GFP, implicating the construct is not expressed in these tissues, i.e. fibroblasts, myofibroblasts, and smooth muscle cells, in contrast to cardiomyocytes (cyan triangle and bottom magnification panel). ITR = Inverted terminal repeat. TropT = Cardiomyocyte specific troponin T promoter. GFP = Green fluorescent protein. PolA = Poly-A tail. H Representative Western blots of in vivo rAAV KD efficiency of the three Igf1r KD hairpins compared to the Ren.713 control hairpin. Hearts were harvested on P5 after rAAV injections on day one after birth and blotted for IGF1R and GAPDH protein levels. Bar graphs indicate mean relative signaling intensity of IGF1R relative to GAPDH ± SD, normalized to Ren.713. Values are normalized to the Ren.713 group. One-way ANOVA with Dunnett’s correction for multiple comparisons was used for statistical analysis in panels A, D, E. Unpaired two-tailed Student’s t-Test were used for statistical analysis in panel H. Full blots are supplied in Supplementary Fig. 3.

Article Snippet: NIH3T3 mouse fibroblasts (Merck KGaA, Darmstadt, Germany) were transduced with the retroviral particles.

Techniques: Knockdown, Expressing, In Vivo, In Vitro, Control, Transduction, Western Blot, Infection, Targeted Gene Expression, Binding Assay, Cell Culture, Luciferase, Imaging, Recombinant, Virus, Plasmid Preparation, Sequencing, Injection, Construct, Cloning, Immunohistochemical staining, Staining, Negative Control, Two Tailed Test

Journal: Gels

Article Title: Self-Assembling Peptide SCIBIOIII Hydrogel for Three-Dimensional Cell Culture That Promotes Wound Healing in Diabetic Mice

doi: 10.3390/gels9040265

Figure Lengend Snippet: Microscopic morphology of NIH3t3 at days 1, 3, and 5 in 2D and 3D cultures. The 2D group did not have peptide added, and the 3D group had 50 µL of SCIBIOIII peptide solution added in each well.

Article Snippet: High glucose DMEM medium with 10% FBS was used to cultivate NIH3t3 cells (Sangon Biotech, Shanghai, China) in a 37 °C incubator with 5% CO 2 .

Techniques:

Journal: Gels

Article Title: Self-Assembling Peptide SCIBIOIII Hydrogel for Three-Dimensional Cell Culture That Promotes Wound Healing in Diabetic Mice

doi: 10.3390/gels9040265

Figure Lengend Snippet: Cytotoxicity analysis of NIH3t3 in SCIBIOIII hydrogels. Calcein-AM staining analysis of NIH3t3 cells cultured in 2D and 3D on days 1, 3, and 5. Live cells are green.

Article Snippet: High glucose DMEM medium with 10% FBS was used to cultivate NIH3t3 cells (Sangon Biotech, Shanghai, China) in a 37 °C incubator with 5% CO 2 .

Techniques: Staining, Cell Culture